MCB 150 Exam 3 !!

proton concentration in each step

*highest proton concentration=lowest pH in lysosome HIGHEST pH / LOWEST CONCENTRATION endocytic vesicle early endosome late endosome active lysosome LOWEST pH / HIGHEST CONCENTRATION

what about proteins destined to work in lysosome?

-2 step process leads to a phosphate group being added onto #6 carbon of a mannose (mannose-6-phosphate group): chemical signal -this zip code says send me to a lysosome -a mannose-6-receptor recognizes the signal and sends it accordingly *but, a functional lysosome does not possess one. so careful

eukaryotic ribosomes

-4 types of rRNA (5S, 5.8S, 18S, and 28S) -about 75 proteins prokaryotic= 70S, 50S & 30S subunit eukaryotic= 80S, 60S & 40S subunit

-where are 45s gene arrays found? how about 5s? who transcribes these? -what are NORS? -is the nucleolus always visible?

-45s gene arrays (will be processed into 5.8, 18, and 28) are found on 5 chromosomes (processed by snorps). RNA polymerase 1 does 45S transcription. 45S gene arrays are found on 5 different human chromosomes (13, 14, 15, 21, and 22) -5s rRNA found on chromosome 1 (not worked on by snorps, not made in nucleolus. RNA polymerase 3 does 5s transcription -nucleolus forms around the arrays of 45S genes, which are called Nucleolar Organizing Regions (NORs). no histones here -if a cell not actively transcribing rRNA, no nucleolus visible total types of rRNA is 6

cycle of myosin movement: 3. cock forward

-ATP in binding site hydrolyzed into ADP+P (ADP+Pi in binding site) -converted chemical energy into hydrolysis of ATP -mechanical energy of cocking lever arm of myosin -goes from cocking back to locking straight

how often do errors occur in replication? what makes replication accurate?

-E. coli=0.04 -average in all organisms is 1 mistake per billion nucleotides -in vitro, 400 mistakes per cell division DNA polymerase and correct BPs shape is most favorable

step 2: recognize the signal (describe this) -what recognizes NLS's -how do they get to NPC

-NPC cannot recognize every NLS, so intermediate called importin helps -importin is a shuttle protein which recognizes NLS's and binds through protein-protein interaction -importin has its own signal** which IS recognized by NLS, and NPC lets in importin with it's associated NLS and protein

go through what needs to happen for RNA molecules to be exported

-RNA molecules cannot physically display a NES because not a protein -bind shuttle proteins to RNA, proteins display NES, shuttle proteins have NES recognized directly by NPC -mRNA entirely covered by protein when it reaches NPC -at least 1 protein shows NES, NES bound by exportin Ran-GTPs, then goes through NPC -on cytosolic side, ATP dependent RNA helicase peels away export related proteins and facilitates binding of protein designed to bind mRNA in cytoplasm

what happens next? -what is GEF

-Ran-GDP must be let back into nucleus -there is a protein specialized to shuttle Ran-GDP (guanine nucleotide exchange factor, or GEF) -GEF brings Ran-GDP back (HE HAS NLS) into nucleus and converts it back into Ran-GTP (cycle complete)

contracted sarcomere

-Z lines are close together, pulled in towards M-line -H-zones disappear/gets smaller -I-band disappear/get smaller -A bands DO NOT change

STARTING NUCLEAR EXPORT NOW. start with the signal

-a protein must have a nuclear export signal, or NES, to go out into cytoplasm from nucleus -NES has abundance of Leu's (Leucine rich)

what signal gets you to the ER? -name and specific name -what does and what doesn't this do? -what if this is your only signal?

-a signal peptide gets you to the ER -specifically, this signal is an AMINO-TERMINAL (N-terminal) signal peptide -this does not attach a protein to a membrane -with only this signal, pushed through translocon entirely and becomes a soluble protein (will be secreted outside cell)

frameshift mutations

-addition or removal of a small number of bases not divisible by three -disrupts the normal reading frame in the mRNA -result: all codons downstream of the change are altered -effect on protein product: usually results in premature appearance of stop codon → truncation

step 3: take the appropriate actions (describe this) -what is Ran? what is its job? -what are its different states associated with?

-after being let in, importin needs to let go of protein -Ran is a protein w/ a nucleotide binding site for GTP or GDP (States are Ran-GTP or Ran-GDP) -Ran-GTP has high affinity for importin, so when importin comes in, gets bound by Ran-GTP -this causes importin to let go of cargo protein, so cargo protein is free in nucleus

how are errors reduced?: mismatch repair

-after replication if proofreading does not catch (post-synthesis) -proteins scan for recently synthesized DNA, looks for mismatches -finds right strand due to chemical marking (methylation) to signify parent/daughter because new DNA gets methylated at adenine -mismatch repair enzymes fix problem in unmethylated (new) strand -once methylated, no way to distinguish parent vs daughter -process: recognize mismatch, remove DNA with incorrect base, resynthesize with older strand as template

"the site of translation determines your fate"- how? -where does translation initiate? -membrane bound vs free cytosolic ribosomes

-all translation initiates at free cytosolic ribosomes -if fully translated on free cytosolic ribosome: stay in cytoplasm, present transit or NLS signal (mitochondria or nucleus), chloroplast, peroxisomes (detoxification with peroxisomal targeting sequence) [ex: Krebs Cycle] -going anywhere else will be made partially attached to rough ER

so, what must happen for additional membrane passes? and how many can we have?

-alternating signal peptides and stop-transfer sequence -can always add a membrane spanning sequence (no limit)

secretion: constitutive

-always present, always produced in equal amounts or at equal rates, regardless of the amount of substrate -no signal needed -digestive enzymes

what's a biparte sequence?

-another NLS -has two parts that are separated, in both, amino acids are + charged/basic *BiP is a NLS which is separated in the middle so it has no ribosomal part, not a ribonucleic protein complex.

actin polymerization -it happens head to tail, but how does it start? -what comes after?

-begins with nucleation, when a G-actin monomer runs into another and collides, eventually building to dimers colliding with other dimers (rate limiting step) -polymerization occurs at both ends and is reversible -once nucleated, make longer polymer through elongation

endocytosis: making lysosomes (receptor-mediated endocytosis) -what is each step of making a lysosome?

-bring in portion of plasma membrane, pinch off to create a new and separate membrane bound compartment, creates early endocytic vesicles -many endocytic vesicles from endocytosis fuse together to create early endosome -early endosome accepts lysosomal enzymes from golgi (vesicles formed from early endosomes and fused with vesicles from trans-golgi carrying hydrolases) to form late endosome -maturation into active lysosome that digests macromolecules *these all have ATP-dependent proton pump

describe this channel

-can be closed to a certain point or opened wide, but cannot be completely closed -if small enough, a molecule can slip through in the closed position

What is N-linked glycosylation?

-carb modification, addition of oligosaccharide tree (group of sugar molecules) to asparagine residue (amino acid)

electrochemical gradient

-chemical=pH difference, electrical= concentration of positive protons -relative to matrix, IMS is + charged. relative to IMS, matrix is - charge because lower conc of _ charged protons -this is source of potential energy

which direction do proteins travel through? how?

-cis-> trans face -each enzyme with its own reaction has its own layer in the golgi (each modification has its own compartment) -different sugars and modifications act as signals/ "molecular zip codes"

what side of the golgi is the cis? trans? -how do you know

-cis: new cisternae, side of golgi that faces nucleus, comes first -trans: old cisternae, side of golgi that faces away from nucleus, these break up look at relative size of vesicles. vesicles on cis size smaller, trans side has larger vesicles. trans side is further from nucleus. RER is continuous with outer membrane of nuclear envelope (RER, look for ribosomes), that will be closer to cis

does all DNA replication happen immediately when S phase starts (and it just takes long), or do some ori's start earlier/later? how do we know?

-do fluorescent dye, use a different color early in S-phase and another late in S-phase -if all replicated at the same time, all would be one color (ex: all red, no blue) -if replicated at different times, both colors would appear (ex: some blue, some red)

what's the point of actin binding proteins (ABP's)?

-don't want it to be recognized for critical concentration, bind protein which keeps it from being incorporated into available concentration -allow for microfilament organization (assembled in dif ways for dif functions)

what is the cytoskeleton? -what is it made of (largest -> smallest)? -what does it do?

-dynamic system of protein fibers that can be reorganized as needed to fit needs of cell -provides physical support and allows for movement -largest= microtubules, middle= intermediate (lamins), smallest= actin/microfilaments (actin and microtubules have directionality) ex of movement: organelles within a cell, sister chromatids, phagocytosis, mitosis, etc can present NLS

chromosomal domains

-each chromosome occupies a distinct territory in nucleus, arranged in organized fashion -nuclei are divided into discrete functional domains that play an important role in regulating gene expression and replication -territory occupied by a DNA molecule (every chromosome have it's territory)

what are the results?

-early replication is scattered throughout nucleus (euchromatin) -late replication is clustered around periphery of nucleus and in pockets in the nucleus (heterochromatin)

why is there a connection between how tight DNA is packed and when it begins to replicate?

-easier to begin DNA replication on DNA that is already unwound (euchromatin)= earlier -tightly wound DNA will be replicated later because it needs to be unraveled= later

how are these brought together?

-enzyme recognizes GPI anchor and protein with specific C-terminal tail -cuts C-terminal tail off protein, hold onto new C-terminus, covalently link it to one of the ethanolamine groups in GPI anchor -covalent interactions all the way down -considered a INTEGRAL MEMBRANE PROTEIN (but they are not embedded in membrane)

what happens after they pass into cytoplasm? -how does exportin let go? -what does GEF do now?

-exportin needs to release the cargo -once GTP is hydrolyzed to GDP, exportin is not attracted and dissociates, releasing cargo protein into cytoplasm -next, exportin returns to nucleus via NPC and Ran-GDP is transported back, GDP released with GTP by the Guanine Nucleotide Excision Factor (GEF)

removal of 3 bases: cystic fibrosis

-faulty protein (CTFR), most cases due to losing a single amino acid in CFTR protein that is very long -causes protein to not fold correctly -Cl- accumulates in cells, causing osmotic balance, water cannot be moved correctly -less hydration outside of cell, mucus layer dehydrates, interfering with breathing/digestion -not weeded out because selective advantage to cholera

nucleotide excision repair

-fixes DNA damage from UV light -removes thymine dimers, uses damaged DNA as template to synthesize new DNA

what are the 'spots' you see? how many replicons in each? how many spots in a human genome?

-foci of replication (cluster of oris), showing various regions where replication is occurring -each replication focus is 200-300 ori's (replicons) (number of foci is NOT equal to # of replicons) -replicon= unit of DNA that comes out of a single ori -would see 100 dots in human genome

cycle of myosin movement: 5. power stroke

-forcible ejection of phosphate group causes "powerstroke" and a stronger actin/myosin bond -returns myosin to cocked back conformation but with new monomer attached -ADP ejected out, cycle restarts with myosin holding nothing

how is the GPI anchor prepared?

-glycolate a phosphatidyl-inositol to get GPI anchor -phosphates and ethanolamine groups make up anchor as well

what's the difference in the golgi of plants?

-golgi is mostly devoted to non-cellulose components that go into the cell wall -called cell wall biosynthesis

cycle of myosin movement: 4. ADP

-headgroup of myosin is lined up with a different monomer in actin filament now -relative to where we began, closer to plus side (barbed) -ADP myosin has actin affinity so it re-attaches to actin -reattachment causes +Pi to eject (only ADP in binding site)

how is the mitochondria streamlined?

-helps give cell energy by helping with oxidative phosphorylation -streamlined b/c delegate tasks to nucleus, mitochondria does NOTHING but o.p, electron transport, and transcription/translation

insertions/deletions: disorders

-hemophilia A caused by insertion -muscular dystrophy caused by deletion -duchenne discovered muscular dystrophy, created ETC, and real (duchenne) vs fake smile

actin monomers (G-actin) -what is it made of? -what does it bind to? -what are its directions? -what are its properties?

-highly folded globular proteins -43 kDa, 375 amino acids -nucleotide binding site for ADP or ATP -has directionality, a plus (barbed) end and a minus (pointed) end WHERE minus has more ADP, plus has more ATP -highly conserved and very abundant, helical ( each subsequent monomer added to the filament is rotated relative to the previous one in a way that does not block the nucleotide binding site)

missense: sickle cell anemia

-his/arg vs. his/asp in active site changes charge of protein -characterized by defective B-globin subunit in hemoglobin protein -distorted cells get stuck in vessels, causing pain/anemia -natural selection has not eliminated because carriers have high-speed turnover rate of RBC, removing malaria (healthier in malaria rich conditions)

critical concentration

-how much G-actin is necessary to be at equilibrium, how much actin do you need for no net growth (stable) -for every one loss, one gained -if above, polymerizing. if below, depolymerizing. if at, no net growth

how can we drop affinity for actin?

-hydrolyze ATP in nucleotide bonding site so ADP is there -ADP bound G-actin has low affinity for other actin and wants to leave, but must wait for G-actin in front of it

how do they get back to cytoplasm? -what is GAP and its job?

-impotin has signal which allows them to be transported in either direction across NPC, so after cargo is let go, gets back across -now importin and Ran-GTP is in cytoplasm, where another protein is found (GAP) -GAP hydrolyzes GTP attached to Ran, and Ran-GDP has low importin affinity, so it lets go

mitochondria ribosomes

-in matrix of mitochondria -only a single piece of rRNA in large subunit and a single piece in small subunit -12S (small) and 16s (large) -carry out translation with these ribosomes -use mitochondrial RNA polymerase (3-5 exo) to transcribe 13 protein coding genes, mRNA will be translated by mitochondrial ribosome (13 genes are transcribed/translated in matrix)

closed state vs open state weight of NPC what ensures the right material is leaving/being let in?

-in most closed state, diameter of 9nm -at most open state, 26nm to accomodate a large ribosomal subunit 125MDa polypeptide chains ensure only material that is supposed to get in nucleus gets in/visa versa

parts of the mitochondria (and label)

-inner and outer membrane -space in-between= intermembrane space -folds of the inner membrane= cristae -internal aqueous environment of the mitochondria= matrix

what is the process of making a multipass transmembrane protein? (in this ex, internal signal first)

-internal signal recognizes by srp, translation paused, complex moved to ER, SRP docks with SRP receptor, let go and signal peptidase folded into translocon with N-terminal facing cytoplasm, C-terminus in lumen (THIS IS SAME) -to make translocon close before translation is finished, must use stop transfer sequence -ribosome continues translation, now both terminus (N and C) facing cytoplasm -for another membrane spanning region (or C terminus in lumen), use signal peptide to open translocon

active transport

-large= >9nm or >40kDa/40,000Da requires energy -energy comes in form of GTP hydrolysis occurring in cytoplasm -most proteins too large to pass through NPC an are energy dependent (proteins, rRNA, etc)

what about the removal of 3 bases?

-loss of one amino acid with the rest of the protein unaffected -not really a frameshift since the reading frame isn't changed

translocations: disorders

-lymphomas and leukemias -philadelphia chromosome

what are the lysosome's tools?

-lysosomal acid hydrolysates -hydrolyze macromolecules in the lysosome -functional at acidic pH's only (maximally active at pH 5) -have multiple types of each for breakdown: proteases and peptidases (proteins), lipases (fats), nucleases (nucleic acids), glycosidases (sugars), phosphatases (phosphate groups), sulfatases (sulfate groups)

nuclear pore complexes -what do they do -does the nuclear envelope allow transport? why? -what structure allows transport?

-main mechanism for transport of materials from 1 side of nuclear envelope to the other -nuclear envelope by itself does not allow transport because it's lipid bilayer has selective permeability -channel in the middle of complex

lysosomes

-major breakdown tool -roughly spherical, not uniform in shape, material being digested inside (what it's degrading defines shape) -toolbox -do degrade themselves eventually

Why are mistakes in transcription/translation not as critical as replication mistakes?

-many copies of RNA produced, synthesizing one mutated gene won't affect blueprint -RNA not heritable over multiple generations (individual protein molecules and RNA's not passed down)

George Palade: Pulse-Chase Experiment

-mark a molecule to detect its path -pulse= mark a 'sample' to watch (feed cell a radioactive isotope to make sample glow) -chase= give no new 'glow' markers, flush with excess of non-radioactive, no new proteins watched -hot= radioactive, cold= non-radioactive

single-pass transmembrane protein

-membrane protein in which the polypeptide chain crosses the lipid bilayer only once -can have N-terminus in cytoplasm and C-terminus in lumen of ER or C-terminus in cytoplasm and N-terminus in lumen of ER

more on the mitochondria

-mitochondria can make all the proteins they need with their 22 types of tRNA -genes for these found in mitochondrial genome -most genes moved over to nucleus (most mitochondrial genes are not coded for in mitochondrial genome)

human mitochondrial genome

-mitochondria have their own DNA and can make some of their own proteins -genome is 16.5kb of DNA (streamlined) -13 protein coding genes (part of electron transport complexes / ATP synthase) -2 rRNA genes (THIS IS THE ONLY THING MITOCHONDRIAL MADE) -22 tRNA genes -ori -1 type of polymerase, does have a transit sequence

how are errors reduced?: DNA proofreading

-molecular backspace key, only adds a new dNTP if the previous is correct, DNA polymerase recognizes mistakes -DNA polymerase with 3-5' exonuclease activity which finds error, removes wrong dNTP on newly synthesized strand, checks template, and replaces in 5-3' direction (works bc geometry incorrect) -reduce by factor of 100

dying for splicing factors

-molecules within snorps. -could be scattered randomly or clustered into regions of specialized function -are clusters of splicing factors -splicing factors are in interchromosomal domains

where is polymerization favored?

-more collisions occur at the plus end -polymerization favored at the barbed end- faster here -depolymerization favored at the pointed end- slower here -if more G-actin than necessary, favors polymerization til back at CC. if below, favors depolymerization until back at CC.

actin is used to promote movement. what other type of protein must be used in conjunction?

-motor protein: myosin -converts chemical energy of ATP into mechanical energy to generate movement -all myosin movement powered by ATP hydrolysis -will get closer to end of their respective cytoskeletal element

class II myosin

-move towards barbed end of actin filaments -the tail part can be attached to transport vesicle or another pair of myosins facing opposite direction

5-10 million ribosomes must be synthesized everytime the cell divides- how do we meet this demand?

-multiple copies of each type of rRNA -280 copies of 5.8, 18, and 28S. 2000 copies of 5S rRNA -arranged in tandem arrays (back to back grouping of identical rRNA) -tandem arrays of genes for 5.8S, 18S, and 28S are transcribed by RNA polymerase I into primary transcripts or pre-mRNAs

how are you allowed in the nucleus? who discovered this? describe. THIS IS ALL NUCLEAR IMPORT BELOW HERE

-must a nuclear localization signal, or NLS -discovered by Dan Kalderon/Alan Smith -rich in positively charged/basic amino acids (lys, arg) -necessary and sufficient to allow protein to enter nucleus -can create NLS when protein folds (proper folding)

how are errors reduced?: methyl-directed mismatch repair (MMR)

-mute-S finds mismatch and says "problem found", but does not know which strand is which. tells complex where to stop after problem -mute-H looks for heavy methylated DNA, if there is a problem, can work together. does not look for mismatches, knows which strand is which. cuts non methylated, removes bases beyond mismatch. exonuclease -mute-L links S and H to form complex -DNA polymerase III does not re-prime, fills in missing gap of DNA then leaves -DNA ligase seals nick -if mismatch repair enzymes missing, error frequency is 100x higher -together, proofreading and mismatch reduce by factor of 100,000

cycle of myosin movement: 1. rigor confirmation

-myosin moving along actin, myosin is latched firmly onto actin and is not letting go -cocked back -rigor mortis: muscles locked into place because no ATP to make myosin let go -nothing in binding site

what must happen along with the removal of the transit sequence? -then, who coats the protein? who folds?

-need to remove presequence and pull protein through matrix -TIM has ability to grab onto protein as its coming through and pull it all the way in -a second set of chaperones coat protein (free floating mitochondrial HSP's) -these chaperones recognize they need to be folded. sometimes, can fold. if they cannot, accompany protein to folding machine

missense: PKU

-newborns screened to prevent with a special diet, caused by single amino acid change -must stop elevated levels of phenylamine to stop mental impairment -in wild-type, phenylamine turned into tyrosine. in mutation, elevated levels of p. is toxic to nerve cells and can't get tyrosine, stunting growth and pigmentation

secretion: regulated

-not constantly present; production is turned on (induced) or turned off (repressed) in response to changes in the substrate concentration -something regulates and tells when it is appropriate to release -insulin

inversions: disorders

-often silent, but can cause infertility

what happens once in matrix? -who removes the transit sequence and how?

-once N-terminus of protein emerges in matrix, transit sequence needs to be removed -removed by special kind of protease called transit peptidase or mitochondrial processing peptidase (MPP) -MPP chops off transit sequence and leaves the rest of the protein, acts as soon as it can

what happens after bound to a transit sequence? -what is the interaction between TIM and TOM? -how is the protein inserted? what happens to chaperones? -what sequence does the protein go in?

-once TOM is bound a transit sequence, TIM and TOM become closer -protein inserted into channel with transit sequence first, and chaperones are peeled off in ATP-dependent manner -protein goes through TOM, and then must pass intermembrane space and go into TIM (requiring energy)

duplications: disorders

-oncogenes activated, becoming cancers -fragile X syndrome and Huntington's are caused by duplications of short, repeated sequences

how is DNA replication organized?

-ori's are brought out to periphery of their territories to allow for clusters of replication machinery to begin replicating from that point -easier to bring DNA to machinery then to move machinery to DNA

multipass transmembrane proteins

-pass through the bilayer several times -have several start-transfer peptides and several stop-transfer peptides

when it's time for a membrane to get larger or replace lost lipids, membrane lipids need to be replenished. how does this happen?

-phospholipids will be synthesized in SER using machinery collected on cytosolic surface -to make new phospholipid, need glycerol, fatty acid tails, phosphate group, and chemical group to be linked to other side of phosphate -all precursors are assembled and integrated by enzyme bound to cytosolic leaflet of SER

what about plants?

-plants lack lysosomes, but have vacuoles that assume the same function -vacuoles also help with osmotic balance and storage

what is the process of making a transmembrane protein with its N-terminus in lumen and C-terminus in cytoplasm?

-present N-terminal signal -N-terminus in cytoplasm, C-terminus faces signal peptidase and is about to get chopped off -new N-terminus of protein in lumen of ER. if no other signal, push amino acids through translocon -if transmembrane, present a stop-transfer sequence, closing translocon -protein ejected into membrane -now, it crosses the lipid bilayer one time -when stop codon encountered, translation stops, subunits dissociate, C-terminus left in cytoplasm *2 signals, 1 chopped off, C-terminus in cytoplasm because ribosome finished protein when already ejected

how is the protein prepared?

-protein prepared as transmembrane protein -N-terminus in lumen of ER (N-terminal signal presented first) -C-terminus in cytoplasm, so had a STS

explain the process of moving to the rough ER

-protein synthesis begins on a free ribosome, ribosome synthesizes ER signal sequence -signal peptide of emerging protein targets translation to RER -signal recognized by the S.R.P (signal recognition particle- ribonucleoprotein complex) -signal sequence binds to SRP, stopping protein synthesis (pauses/blocks translation) -ribosome, signal sequence, and SRP move into rough ER -GTP binds SRP to SRP receptor. once receptor and SRP connect, protein gets inserted because translocon opens -once GTP hydrolyzed, SRP released and protein synthesis continues in translocon

process of mitochondrial export -what happens first -what stops it from folding -who recognizes transit signal

-protein translated by free cytosolic ribosome displays transit sequence at N-terminus -bound by cytosolic chaperones which stop from folding in cytoplasm -binding proteins/chaperones are 'cytosolic Hsp70s' -transit signal recognized by receptor protein in TOM complex

how does this change the original protein? (under microscope) does this happen often? what must happen before it moves to the next location?

-protein with 14-mer, can be multiply glycosylated, virtually every protein gets in multiple locations -may make proteins look "fuzzy" under microscope -want to make 14-mer into 11-mer, lose 3 glucose residues which is sign of maturation that shows protein is ready to move to next location

Golgi apparatus

-proteins travel in vesicles that bud off from the ER and fuse with cis G.A (dumping cargo) -translated/folded/modified proteins are sent to GA for additional processing and sorting -system of membranes that does final protein processing -sorting to know where to send (finding final destination- get ready for shipping)

what are examples of where what is made on membrane bound ribosomes will end up?

-proteins working in ER (ER-resident protein) -golgi resident proteins -lysosome or endosome resident proteins

H-zone

-region at the center of an A band -made up of myosin only -gets shorter (and may disappear) during muscle contraction. -brighter part of A-band

what's the D-loop?

-regulatory region of non-coding DNA in mitochondrial genome -ori is in D-loop and multiple copies of genome in matrix

base substitutions: definition, where they can occur, and types

-replace one base pair with another -can happen anywhere in genome (intron, space, tRNA, protein-coding) transition (base of same family) or transversions (other family of bases)

describe the structure of NPCs

-ring structure on cytoplasmic and nuclear side with 8 fold symmetry, these are protein complexes -30-50 types of polypeptides -large (30x ribosome size) -central channel has ring acts as diaphragm to open and close -on nuclear ring, basket structure. on cytoplasmic ring, protein filaments

how do these exporter complex proteins get back into nucleus? -why do we sometimes see proteins in either side without the appropriate signal?

-show both NLS and NES -some proteins without NLS/NES pair up with molecules pair up with molecule with targeting signal (partner with appropriate signal)

what recognizes the NES? what happens next? what's exportin's job?

-signal is recognized by exportin, and NPC recognizes exportin's own signal** -protein needing to be exported displays NES which will be bound by exportin (but exportin has low affinity for this protein) -exportin has high affinity for Ran-GTP, and Ran-GTP binding induces exportin to bind to cargo -protein complex of 3 approaches NPC and is allowed to pass into cytoplasm *remember, Ran-GTP makes exportin bind to cargo protein

internal signal sequence

-signal peptide that is somewhere other than the N-terminus -remains as an integral membrane protein (is not cleaved by a signal peptidase)

process of N-linked glycosylation -what makes the signal -what is dolichol? tree? -how do they come together?

-signal: 3 amino acids (1 asparagine with nitrogen) make signal -covalent addition of sugar group onto nitrogen within asparagine -must build oligosaccharide tree, begins with addition of a few sugars onto lipid soluble molecule (dolichol) -dolichol is oddly shaped lipid which is platform that gets sugar molecules attached -put together oligosaccharide tree with 14 different monosaccharides (14-mer), build tree of sugars on dolichol platform -there is a enzyme responsible for recognizing asparagine residue and prepared 14-mer. it cuts 14-mer off dolichol platform and covalently links it to asparagine in 3 amino acid sequence

how is the protein drawn to TIM?

-since transit sequence has a + charge, want to be in the - matrix. thus, it is drawn to TIM out of IMS -negative matrix attracts basic amino acids in transit sequence and do not have to use ATP

passive diffusion

-small= <9nm or <20kDa/20,000Da can pass freely in NPC, going in terms of concentration (high to low) -no external energy source needed

what is the process of making a transmembrane protein with it's C-terminus in the lumen and N-terminus in the cytoplasm?

-start by showing internal signal sequence -treated the same: internal signal sequence recognized by srp, ribosomes pause -move to ER, dock with SRP receptor, signal peptide is folded into translocon -N-terminal most amino acid faces cytoplasm, C-terminal most faces lumen -DO NOT cut internal signal sequence, signal peptidase will leave alone -protein continues to be threaded into lumen of ER, but N-terminus stays in cytoplasm -translation stops when stop codon reached (translation terminates, subunits dissociate, etc) -translocon closes shut when this happens, squeezing out the internal signal sequence, which is now ejected into membrane

this would cause an imbalance in the # of phospholipids from one leaflet to another. how is this solved?

-take 1/2 phospholipids made and move to other leaflet -transverse diffusion is energy dependent, need an enzyme -enzyme= flippase

next in the process -what is the channel the protein will move through? -describe the two complexes -when do they open? which directions do they go?

-the channel that the unfolded protein will move through is called the translocon/translocator (includes a number of polypeptides on outer and inner membrane) -translocator of outer membrane= TOM complex (recognizes transit sequence) -translocator of inner membrane= TIM complex -TOM and TIM close all the way unless a protein is being imported into the mitochondria -proteins do NOT get out through TIM/TOM (if no tim, would end up in IMS)

what is the secondary signal which are recognized as "insertion signals" for the ER? (necessary for transmembrane proteins?)

-the secondary signal is a stop-transfer sequence -stretch of hydrophobic amino acids (like signal peptide) -gives instructions to translocon to eject protein out into the membrane -unfolded in translocon

cycle of myosin movement: 2. myosin letting go

-to make myosin let go, need ATP -headgroup has nucleotide binding site for ATP (ATP in binding site) -once ATP binds, myosin lets go of actin

where are mitochondrial genes transcribed/translated, and how are they transported?

-transcribed in nucleus, translated in cytoplasm -once protein is translated by free cytosolic ribosome, must be localized into mitochondria using signals

what happens after SRP is released?

-translation resumes in translocon -signal sequence cleaved off by signal peptidase as protein gets longer and moves from cytosol to lumen of ER -completed protein is released into lumen, translocon pore/channel closes -process: co-translational insertion *SRP can bind to ribosome, SRP receptor, and internal sequence

how can actin/myosin be used to move transport vesicles inside the cell?

-type 1 myosins hold vesicle -need to be other myosin's around, because as soon as one lets go, need to still keep vesicle attached to actin and moving -MANY type 1 myosins on surface of vesicle -after 1 powerstrokes, next grabs on -vesicle appears to be rolling along actin filament

does the ribosome care about this?

-unaware whether translocon is open or closed -only a stop codon will cause a ribosome to stop making a protein!! will continue making a protein even after protein is ejected into membrane

is transcription/pre-mRNA processing organized or random? how can we tell?

-use fluorescence -stain with dye specific for chromatin, so it binds and glows. more light= more chromatin -this shows you chromosomal territories (more white, more chromatin) vs interchromosomal domain (darker, less chromatin) -nucleoli appear dark bc dye can't reach chromatin

how could we see if DNA replication is random or organized?

-use fluorescently labeled dNTPs -find each ori, recognize and unwind. each bubble unwinds, allow replication to begin using fluorescently labeled nucleotides -see where DNA replication is initiating (location of fluorescence tells you where DNA replication is occurring)

what is the rule of the golgi?

-what starts cytosolic stays cystolic -what starts lumenel stays lumenel or could be exposed to extracellular space -what's in the lumen will never touch the cytoplasm

filament sliding how did we visualize this??

-when myosin can't move, actin will, which causes filament sliding -pulls ends of actin filaments towards each other, meeting in middle myosin slide experiment, fluorescent actin. if supply ATP, myosin will walk to barbed end. since myosin can't move, actin filaments will, and we can see glowing filaments walking

uncontracted sarcomere

-wider I-bands that is just actin -wider H-zones that is just myosin

in electron microscopy, what distinct regions of the nucleolus can be distinguished?

1. DNA molecules in pouches- fibrillar region/center [rDNA here] 2. black on the periphery of fibrillar region- dense fibrillar region (where transcription factors are) [pre-rRNA here] 3. granular zone- everything else [pre-ribosomal particles here] we then get a ribosomal subunit transcription, processing of rRNA, and subunit formation happens in these regions/zones

cytokinesis (part of M phase, cytoplasmic division): how can we use actin/myosin? explain.

1. build ring of actin filaments around interior portion of cytosolic leaflet of the plasma membrane (inside) 2. once contractile actin ring formed, associate type 2 myosin with it 3. all barbed ends face on direction, all myosins move towards barbed end 4. start cinching belt, length of contractile ring changes 5. movements of myosin and the depolymerization of actin leads to division of cytoplasm into 2 daughter cells 6. back to interphase (g1)

using existing lysosomes: phagocytosis

1. create phagosome when a smaller cell/particle is surrounded 2. delivery to lysosome (fuse with lysosome) 3. phagolysosome complex formed 4. degrade what is inside phagosome, material digested 5. cystol for recycling

using existing lysosomes: autophagy

1. detection of large structure/organelle that needs to be recycled! 2. phagosome formation 3. delivery to lysosome and digestion 4. small molecules recycled *decompose damaged organelles to reuse organic monomers- self-eating 5. cystol for recycling

why can't we cut the internal signal sequence out?

1. it's supposed to now represent a membrane spanning region 2. can't afford to lose the amino acids on the other side

process of cell crawling

1. push out leading edge of cell (lamelopod) through polymerization of actin network 2. attach extended leading edge to substratum (anchor actin bundles) through specialized adhesion sites 3. myosins grab onto actin filaments and start contracting, moving entire cell body forward 4. retract trailing edge, detach cell from point of attachment in back 5. cytoplasm rejoins rest of cell

what are the "caps" on the pointed ends of actin filaments?

ABPs that stop actin from polymerizing/depolymerizing

what can actin and myosin do for muscles?

Actin and Myosin cause muscle contraction in sarcomeres in a skeletal muscle fiber

where is SER abundant?

Cells active in lipid metabolism -cholesterol is lipid and precursor for steroid hormones. large amount of SER found in steroid producing cells like testes/ovaries. the more SER, the more area to accommodate enzymes that convert cholesterol to hormones Liver cells -detoxification. metabolize potentially harmful lipid-soluble compounds into water soluble compounds that can be removed in urine. for example, barbiturates get moved to liver where SES lumen enzymes convert them to excrete,

what functions are carried out in the nucleus?

DNA replication, transcription, ribosomal subunit assembly. all regions of function in the nucleus that are very organized

what proteins are functioning in the nucleus?

DNA/RNA polymerase, transcription factors, histone proteins, ribosomal proteins, snoRPs, nuclear lamins

cell cycle: each stage and what happens

M-PHASE mitosis, one cell divided into two daughter cells, then go into G1 INTERPHASE (longest phase, 1/3 of cell cycle) G1= 1st gap: cell growing, building up nutrient base, checking for DNA damage, prepare for replication S= synthesis: cell processes don't stop, but add replication (G1+replication) G2= 2nd gap: prepare for M phase, fix DNA problems

are ALL barbed end directed?

Most (nearly 100%), BUT NOT ALL. 1-2 classes of myosins are pointed-end directed

what if a mutation slips through? (categories)

Point mutations base substitutions (same sense, missense, nonsense, silent) and frameshift Chromosomal level insertion, deletion, inversion, duplication, translocations

predicting amino acid changes from base substitutions

Possible Changes: • same box- stay in same box if change in 3rd position • same row across- stay in same row across if change in 2nd position • same position vertically- stay in same relative position vertically if change in 1st

what molecules are functioning in the nucleolus?

RNA polymerase I, snoRPs, ribosomal proteins

secretory pathway

Rough ER -> Golgi -> Secretory Vesicles -> Exterior (Other Destinations) -protein enters rough ER while being synthesized by ribosome -protein exits ER inside a vesicle, travels to cis face of golgi -protein enters golgi and is processed, then exits through secretory vesicles to move to plasma membrane -protein secreted from cell

lumen of ER

The space between the inner and outer nuclear membrane is continuous with this, inside of ER

where are the actin filaments? myosin? what are the 4 steps? how is this possible?

actin filaments fill entire cell, contractile myosin fibers located in cell body extension, adhesion, translocation, de-adhesion due to polymerization and depolymerization of actin filaments + movement of myosin

structure of nucleus: nucleoplasm

aqueous compartment inside the nuclear envelope, nuclear counterpart to the cytoplasm

what is the point of the structures on each side?

basket aids in recognition of RNA molecules ready for export, cytoplasmic filaments aid in the recognition of proteins that need to be imported

structure of nucleus: chromatin fibers

beads on string, DNA and protein together

what do you see before, during, and after the pulse? what did regions or randomly distributed show? what were the results?

before: lots of protein, no glow/radioactivity, impossible to follow during: actively incorporating radioactive amino acids, new proteins glow, old proteins do not after: new proteins non-radioactive, pulsed still radioactive- can finally track -start where synthesis occurs either clustered in regions (membrane bound) or randomly distributed (free cytosolic). -track path after to show overall path of proteins in marks of time: found the secretory pathway !

bulk flow vs retrieval

bulk flow: bulk flow of material through secretory pathway, RER -> cis side of GA -> trans face -> outside retrieval: backwards process, occurs because you were sent there by accident (ex: ER resident protein accidently packaged in transport vesicle and sent to golgi) and because even proteins supposed to be working in ER are not finished. have final processing and then have ER retention signal to go back

how is directionality determined?

by 1st stop signal a. if first signal is N-terminal, N-terminus in lumen b. if first signal is internal signal, N-terminus in cytoplasm

how can you tell these methods of secretion apart?

by size of vesicle. constitutive is smaller because it is always being used up, regulated is bigger because it is being packed away/stored

how was chromosome painting used to further research on domains?

can paint chromosomes different colors (ex: chr 1 red, chr 2 blue, with dif dyes for each). when done on interphase chromosomes, shows that there is no overlap of colors- domains are organized, interphase chromosomes occupy their own territories

what are actin and myosin responsible for?

cell division, cell crawling, vesicle movement, and muscle contraction

conventional vs unconventional myosin

conventional: capable of forming thick filaments and spend most of cycle not attached to actin unconventional: not capable of forming thick filaments, headgroups spent much more time bound to actin (Type 1) *both barbed end directed

A-band

dark area; extends the width of the thick filaments, but has overlapping actin. on both sides of M-line.

structure of nucleus: nucleolus

densely staining region within the nucleus, produces ribosomes

how do molecules that can't pass through lipid bilayer get from one side of the nuclear envelope to other?

depends on size

cleavage furrow

drawing in of the plasma membrane

what do all mitochondrial proteins relate to?

electron transport or oxidative phosphorylation *necessary for functional ETC and ATP synthase, but not sufficient

chromosome painting

extension of FISH. uses multiple probes, each specific for a different region of a particular chromosome. can light up entire length of chromosomes, not just parts.

micrope: fluorescence vs electron

fluorescence= brighter is more, electron= darker is more

sarcomere

functional, repeating units of contraction in skeletal muscle tissue (myofibril). spans distance from dark line in one white band to dark line of the next

what is a mutation? gene+ vs gene-

heritable change in DNA if +, wild type. if -, mutated

how does gene density relate to replication?

high gene density will be replicated earlier in S-phase (more likely to exist as euchromatin, already unwound) low gene density will be replicated later in S-phase (more likely to exist as heterochromatin, packed up and moved to periphery)- darker in EM

2 hypothesis for DNA replication

if unorganized, light red color diffused throughout nucleus because various ori's are firing randomly if organized, clusters of DNA replication will be present (this is what happened, proving it is organized)

structure of nucleus: perinuclear space

in between the outer and inner membrane. region around nucleus just outside inner membrane, while inside outer membrane

chromosomal level mutations: types and definitions

insertion/deletion: adds or deletes large chunk of DNA (meaning thousands or more chunks), DNA was not part of this chromosome before duplication: 2 copies of a large chunk of DNA- replicated copy of region of genome that was a part of the chromosome inversion: flips large chunks of DNA translocation: swaps regions of nonhomologous chromosomes, NOT increasing genetic variation

nuclear lamina

just inside inner membrane, system of ropelike protein fibers that provide point of attachment for heterochromatin and give nucleus shape/support (would collapse without)

what do NPCs look like in an electron micrograph?

large, numerous symmetrical flower like shapes with 8 fold symmetry with a channel through the middle

what will never change? what moves?

length of actin and myosin- these will never change sizes, only the overlap myosin is stationary other than headgroups moving back and forth, relative movement comes from actin

I-band

light band with thin (actin) filaments only, no overlapping myosin

what do nuclear domains provide for?

localized regions of functions for the activities that occur in the nucleus

modifications that continue in the G.A from the RER how will packages end up at their desired location?

mannose, galactose, fructose, sialic acid, N-acetylglucosamine signals

structure of nucleus: nuclear pore complex (NPC)

many inside of the nucleus, this is how larger molecules are transported in and out of the nucleus

microvilli and actin bundles

microvilli increase surface area 10-20x, anyone of these microvilli are actin filaments ACTIN BUNDLES

M-line

middle of sarcomere, also the middle of the A band. myosin is anchored here

categories of base substitutions and consequences

missense (one amino acid specifying codon gets changed to a different amino acid specifying codon)- effect varies. if amino acid changes did not alter folding/active site/not critical for function or amino acid that replaces it assumes the same role, can be some or full functioning. if important to folding/activity, inactive nonsense (one amino acid specifying codon gets changed to a stop codon, truncating protein)- premature termination, protein truncated and usually inactive/null phenotype samesense (changed to a different codon specifying same amino acid, silent)- no effect, due to redundancy of genetic code

endosymbiotic theory

mitochondria are evolutionary descendent of ancestral prokaryotic cell, larger cell engulfed the smaller cell because it conferred a selective advantage

what is the signal for mitochondrial export? -what is it made of?

mitochondrial presequence or TRANSIT sequence -peptide sequence rich in basic amino acids in N-terminus of proteins -removing it does not require energy

cell crawling

movement of a cell along its substratum (whatever a cell is resting on)

muscle cell

multinucleated cell that arose from precursor myofibrils

muscle organization

muscles -> muscle tissue -> bundle of muscle fibers (many cells) -> muscle cell (one cell, many myofibrils, many nuclei) -> myofibril (many sarcomeres) -> sarcomeres

muscle contraction

myosin pulling actin filaments into middle of sarcomere, increasing the overlap between thin and thick filaments

why has there been chromatin pushed out into interchromosomal domain?

needs to work on that region of DNA, find machinery to do transcription/processing (put DNA where machinery is)

what folds these proteins into 3d shapes? what are the two things that can happen to the proteins now?

new proteins are folded by chaperones once they are inside rough ER or inserted in membranes 1. completely released into ER lumen- proteins that will be secreted/exported to outside cell 2. need to be integrated into membrane (transmembrane proteins)

structure of nucleus: outer membrane of nuclear envelope

nuclear envelope is double lipid bilayer structure, this is side facing cytoplasm

structure of nucleus: inner membrane of nuclear envelope

nuclear envelope is double lipid bilayer structure, this is side facing nucleoplasm

what did this show?

nucleus is divided into domains that play important roles in how functions are carried out.

gene density

number of genes in a given length of DNA (ex: if 2 pieces of DNA are 100bp, but one has 15 genes and the other has 1 gene, the first is more 'dense')

we are about to discuss movement in/out of the nucleus through the NPC's. first, let's review -how to differentiate between outer and inner membrane -what is the perinuclear space?

outer membrane faces cytoplasm, inner membrane faces nucleoplasm aqueous env in between membranes, continuous with interior of ER

where do NPCs and ribosomes attach to? how do we interpret an electron micrograph?

outer membrane of nuclear envelope if densely stained spheres attached to one side, they are ribosomes, and this is the outer membrane. use this information to label cytoplasmic side vs nuclear side. on nuclear side, very darkly stained clumps are heterochromatin, ribs=lamin proteins, meshwork of proteins is lamina

what is the reason for GPI anchored proteins?

present it to outside world, protein exposed to extracellular face

step 1: present the signal what needs to travel into nucleus?

present the signal (NLS) rRNA, mRNA, dNTPs, proteins, TFs

what are proteases? peptidases?

proteases are enzymes which degrade peptide bonds, peptidases are a subset which remove some amino acids from the end of a protein

chaperonin complex

protein folding machine, complex of individual chaperones. closes to fold protein with no external influence. once folded, complex opens, folded protein let out, and protein can now be used in matrix -opening is final energy dependent step which uses energy source (ATP) -this gives you 1 of 95% proteins not made in mitochondria into mitochondria -finishes mitochondrial localization

structure of nucleus: nuclear lamina

provides point of attachment for heterochromatin, gives nucleus shape and structure, part of cytoskeleton

why do we need to export proteins? -can you display NLS and NES?

ribosomal subunits. had to import ribosomal proteins, send them to nucleolus, get them assembled into pre-ribosomal subunits, then export them. at least 1 ribosomal protein in a subunit must display NES -can display NLS and NES- ribosomal proteins

the nucleolus

ribosome production factory. site of rRNA synthesis, rRNA processing, and assembly of ribosomal subunits

Z-disc

separates the sarcomeres from each other, dark line in I-band. actin is anchored here, anchor + side of each actin

what targets translation to the RER? -Dobberstein and Blobel

signal hypothesis: there is a specific amino acid sequence that targets proteins to the ER

what modifications occur in the RER?

signal peptide removal, chaperone assisted folding, disulfide bond formation, N-linked glycosylation

single cell organisms vs multi-cellular

single cell= all daughter cells have mutation multi-cellular= mutations somatic or germline where somatic is passed to all daughter cells in area, germline is passed to new organism

myofibril

small fiber of repeating units of actin and myosin, make up most of muscle fiber

what did Carl Rabl propose?

solved the question of what the organization of the nucleus was like during interphase. proposed that each chromosome occupies a distinct (its own) territory in nucleus, little overlap in interphase nucleus

interchromosomal domains

space inbetween/around chromosomal domains, their own territory. molecules in this domain carries out functions.

smooth ER: structure and functions

structure: interconnected tubes, no ribosomes functions: lipid bilayer synthesis, detoxification, storage, carb metabolism. cytoplasmic

rough ER: structure and 3 functions

structure: series of flattened pancakes with ribosomes imbedded functions: protein secretion, synthesis of membrane proteins, protein processing (add sugars/lipids/etc) continuous with smooth ER and perinuclear space

what if areas are exposed behind lamina?

they are polar head groups of phospholipids exposed in one leaflet of inner membrane of nuclear envelope

thin filaments vs thick filaments

thick: myosin thin: f-actin

what is alcohol tolerance?

tolerance rising over time due to the smooth ER being extended to accommodate more enzymes involved in detox of lipid soluble -> water soluble (higher capacity to removal ethanol= more alcohol needed)

how can DNA sequences be changed? which of these accounts for the most mutations

uncorrected mistakes in replication, chemical mutagens, high-intensity radiation (x-rays, UV, etc.) most= mistakes in replication

Fluorescence in situ hybridization (FISH)- goal and process

used to visualize and identify chromosomes and identify genes (find small region of chromosome) isolate cells, immobilize on glass slide, crack cells open and get rid of non-DNA parts. denature leftover DNA, allowing us to hybridize things onto it. then, create probe molecule complementary to target sequence that is detectable when it binds (fluorescent). look under microscope for what is glowing.

what about 20-40kDa in size?

will eventually pass through, do not need to be transported, but very slow

how do you create a lipid molecule covalently linked to a protein (lipid-linked protein)? -where does this occur? -is it common? -what other modifications will they usually have?

with a glycosyl phosphatidyl anchor (GPI anchor) -occurs in RER -only happens to a very small subset of specific proteins -most were previously glycosylated -lumenal surface of ER

was Rabl correct?

yes, proven by studies of polytene chromosomes in Drosophila. since the chromosomes do not separate and there are so many identical copies are linked together, they are visible under light microscopy. interphase chromosomes do occupy their own territory, nucleus is organized

is transcription of rRNA genes effective?

yes, very efficient. forms Christmas Tree formations where ornaments are ribosomal proteins/snorps

can the GA help with further lipid synthesis? how? -ceramine, and what are the effect of enzyme A vs B in golgi

yes. site for further lipid processing. -ceramine is made in SER. after its made, transported to GA -if enzyme A takes, adds phosphate group= creates phospholipid. ceramine in SER -> golgi with enzyme A -> sphingomyelin -if enzyme B takes, add carbohydrate, creating glycolipid